Project Summary (as submitted in orginal application) Most eukaryotic pre-mRNAs, especially in metazoans, are alternatively spliced to generate multiple mRNAs and proteins. Given the importance of alternative splicing in regulating gene expression and enhancing the diversity of the proteome, it is essential to understand the mechanisms of splicing and how alternative splicing is regulated. In this project, we will study three unusual types of splicing in Drosophila. The Down Syndrome Cell Adhesion Molecule 1 (Dscam1) gene is the most extensively alternatively spliced gene know. Dscam1 contains 115 exons, 95 of which are alternatively spliced and has the potential to generate 38,016 different mRNA and protein isoforms. We will study how probabilistic splicing of Dscam1 is achieved and how it contributes to determining cell identity, a critical process involved in determining the specificity of neural wiring. The longitidunals lacking (lola) and modifier of mdg4 (mod(mdg4)) genes are the best examples of genes that undergo trans-splicing ? a process by which exons from different pre-mRNAs are spliced together to generate a protein-coding mRNAs. We will study how trans-splicing occurs and the role of homologous chromosome pairing in this process. How very long introns are efficiently spliced has long been a mystery. Seventeen years ago, it was shown that one long Drosophila is removed in a progressive, stepwise fashion by a process called recursive splicing. However, until now it was not known how widespread this phenomenon occurred. We recently identified nearly 200 instances of recursive splicing in Drosophila and that it also occurs in humans. We will focus on elucidating the mechanisms and functions of this unusual process. We will also work with collaborators to study how bacteria and archaea become resistant to newly encountered viruses by means of the CRISPR pathway and the determinants and regulators or RNA turnover on a genome-wide scale. All of these problems will be addressed using a wide variety of cutting edge approaches we have applied or developed including reporter genes to facilitate visualizing splicing in single neuron resolution, single cell RNA-Seq, nanopore sequencing, RNAi or CRISPR screens, BAC recombineering, Drosophila genetics, and computational genomics. We will also continue to develop additional innovative approaches to address these issues as needed or as opportunities arise due to technical advances in the field.